Camostat Research Articles

Type II transmembrane serine protease Matriptase is a mediator of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder which remains refractory to therapies. Recently, type II transmembrane serine proteases have emerged as key actors in pathophysiology, by proteolysis of cell surface receptors and/or cell environment, thereby orchestrating cell crosstalks. Among them, deregulation of the matriptase contributes to various proliferative disorders. Hence, we sought to investigate the role of matriptase in pulmlonary fibrosis. Matriptase expression and activity were analyzed in human lung material from controls and IPF patients. Human fibroblasts were stimulated with recombinant matriptase and the activation of key signaling pathways involved in IPF was analyzed. Using the murine model of bleomycin-induced pulmonary fibrosis, mice were administrated for 14 days with 0 or 0.5mg of camostat mesylate, a matriptase inhibitor, and the markers of tissue fibrosis in lung homogenate, and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) of the animals were assessed. We show that matriptase is upregulated in the lung and BALF of IPF patients compared to controls. In fibroblasts, matriptase induced the activation of p42/44, Akt, and Smad2/3 pathways. Finally, camostat administration in bleomycin-treated mice led to a significant decrease in mortality (39.3% versus 60.4% in bleomycin-treated group). Fibronectin and collagen expression, and inflammatory cell influx, were not significantly different from saline-treated animals in the camostat group.Taken together, matriptase seems instrumental in pulmonary fibrosis, and camostat mesylat could be a promising target for thetreatment of IPF.

LAP degradation product reflects plasma kallikrein-dependent TGF-β activation in patients with hepatic fibrosis.

Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-β plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-β is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-β must be released from the latent complex. Several proteases activate latent TGF-β by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-β during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients.Here, we report that PLK cleaves LAP between R58 and L59 residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients.The N-terminal side LAP-DP ending at R58 (R58 LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L59 (L59 LAP-DP) was not detectable. The R58 LAP-DP was seen mostly in α-smooth muscle actin-positive activated stellate cells.These data suggest for the first time that the occurrence of a PLK-dependent TGF-β activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-β activation in fibrotic liver both in animal models and in patients.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-221) contains supplementary material, which is available to authorized users.

Successful Hemodialysis Initiation in a Patient with Chronic Disseminated Intravascular Coagulation

We report a 72-year-old man with end-stage renal disease due to chronic glomerulonephritis and chronic disseminated intravascular coagulation (DIC) caused by aortic aneurysm. He had had graft replacement for abdominal aortic aneurysm in 2012 at another hospital. He indicated development of thoracic aortic aneurysm, which developed chronic DIC with much subcutaneous hemorrhage. His renal function gradually decreased and we planned hemodialysis initiation. We administered camostat mesilate and platelet concentrate transfusion to treat hemorrhagic tendency before the arteriovenus fistula in May 2014. After the operation, we added tranexamic acid because of difficulty in hemostasis. About 1 month later, the patient reported with hemorrhage from the nose and his renal function was exacerbated. He was initiated into hemodialysis with transfusion of red blood cells and fresh frozen plasma. We raised the dose of camostat mesilate and adjusted the dose of anticoagulant with reference to activated clotting time. He was successfully initiated into dialysis without major trouble.

The antifibrotic effect of a serine protease inhibitor in the kidney

Interstitial fibrosis is a final common pathway for the progression of chronic kidney diseases. Activated fibroblasts have an extremely important role in the progression of renal fibrosis, and transforming growth factor (TGF)-β₁ is a major activator of fibroblasts. Since previous reports have indicated that serine protease inhibitors have a potential to inhibit TGF-β₁ signaling in vitro, we hypothesized that a synthetic serine protease inhibitor, camostat mesilate (CM), could slow the progression of renal fibrosis. TGF-β₁ markedly increased the phosphorylation of TGF-β type I receptor, ERK 1/2, and Smad2/3 and the levels of profibrotic markers, such as α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1, in renal fibroblasts (NRK-49F cells), and they were all significantly reduced by CM. In protocol 1, 8-wk-old male Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO) and were concurrently treated with a slow-release pellet of CM or vehicle for 14 days. Protocol 2 was similar to protocol 1 except that CM was administered 7 days after UUO. CM substantially improved renal fibrosis as determined by sirius red staining, collagen expression, and hydroxyproline levels. The phosphorylation of ERK1/2 and Smad2/3 and the levels of α-SMA, CTGF, promatrix metalloproteinase-2, and matrix metalloproteinase-2 were substantially increased by UUO, and they were all significantly attenuated by CM. These antifibrotic effects of CM were also observed in protocol 2. Our present results suggest the possibility that CM might represent a new class of therapeutic drugs for the treatment of renal fibrosis through the suppression of TGF-β₁ signaling.

Mediastinal Extension of a Pancreatic Pseudocyst: A Rare Cause of Dysphagia in the Setting of Pancreatitis

Introduction: Pseudocysts are a common complication of acute or chronic pancreatitis and are also seen following pancreatic trauma. However, mediastinal extension of a pancreatic pseudocyst is rare. Case: A 40-year-old man was admitted to our hospital with progressive dysphagia and vomiting after meals for 4 days. He had a previous admission for acute alcoholic pancreatitis with pancreatic pleural effusion a year before. After the previous discharge, he was not able to quit alcohol consumption completely after his temporary abstinence. His medications included camostat mesilate and rabeprazole sodium. However, he stopped taking his medications and consumed alcohol frequently over the past 4 months. Chest roentgenogram revealed a distinctly defined retrocardiac mass in the posteroinferior mediastinum. Upper gastrointestinal endoscopy revealed stasis at the lower end of the esophagus, which was compressed extrinsically. Computed tomography scan demonstrated a pancreatic pseudocyst arising from the tail of the pancreas and extending through the diaphragmatic hiatus into the posteroinferior mediastinum. The esophagus was compressed extrinsically by the pseudocyst, which resulted in dysphagia. Treatment consisted of abstinence, fasting, and fluid replacement. His condition improved rapidly and dysphagia also resolved. He left the hospital 10 days after admission and was asymptomatic. Discussion: The mediastinum is a rare site for pancreatic pseudocysts. A mass in this site can cause anterior and lateral displacement of the esophagus and the stomach, which may result in dysphagia. The ideal management for mediastinal pseudocyst is controversial and depends on the underlying etiology, severity of symptoms, ductal anatomy, size of the pseudocyst and expertise available. Successful medical treatments such as somatostatin analogues, total parenteral nutrition, and watchful waiting for spontaneous regression have been previously reported. Pseudocyst internal or external drainage techniques have also been described as being effective, and can involve percutaneous external drainage, surgery such as cystogastrostomy, and endoscopic internal drainage such as transpapillary and transgastric techniques. Our patient was treated with conservative medical management based on his mild symptoms and a spontaneous resolution of the pseudocyst was obtained. Conclusion: Mediastinal pancreatic pseudocyst as a cause of dysphagia is very rare. As the condition can be life threatening, gastroenterologists should be aware of this atypical complication of pancreatitis, although it is rare.

Effect of a serine protease inhibitor on the progression of chronic renal failure

The number of the chronic renal failure (CRF) patients is increasing explosively. Hypertension, proteinuria, inflammation, fibrosis, and oxidative stress are intertwined in a complicated manner that leads to the progression of CRF. However, the therapeutic strategies to delay its progression are limited. Since serine proteases are involved in many processes that contribute to these risk factors, we investigated the effects of a synthetic serine protease inhibitor, camostat mesilate (CM), on the progression of CRF in 5/6 nephrectomized (Nx) rats. Eighteen male Sprague-Dawley rats were divided into three groups: a sham-operated group (n = 6), a vehicle-treated Nx group (n = 6), and a CM-treated Nx group (n = 6). Following the 9-wk study period, both proteinuria and serum creatinine levels were substantially increased in the vehicle-treated Nx group, and treatment with CM significantly reduced proteinuria and serum creatinine levels. The levels of podocyte-associated proteins in glomeruli, such as nephrin and synaptopodin, were markedly decreased by 5/6 nephrectomy, and this was significantly ameliorated by CM. CM also suppressed the levels of inflammatory and fibrotic marker mRNAs including transforming growth factor-β1, TNF-α, collagen types I, III, and IV, and reduced glomerulosclerosis, glomerular hypertrophy, and interstitial fibrosis in histological studies. Furthermore, CM decreased the expression of NADPH oxidase component mRNAs, as well as reactive oxygen species generation and advanced oxidative protein product levels. Our present results strongly suggest the possibility that CM could be a useful therapeutic agent against the progression of CRF.

In vivo contribution of serine proteases to the proteolytic activation of γENaC in aldosterone-infused rats

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.

Proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension

Proteases are involved in numerous essential biological processes including blood clotting, controlled cell death, and tissue differentiation. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as a potential regulator of the epithelial sodium channel (ENaC) function in the kidney, lung, and airways. ENaC is composed of three homologous subunits α, β, and, γ. The dual cleavage of α subunit by furin and γ subunit by prostasin and furin releases inhibitory segments from ENaC, leading to the channel activation. Protease nexin-1, an endogenous prostasin inhibitor, inhibits ENaC activity through the suppression of prostasin activity, strongly suggesting the possibility that a coordinated regulation of serine proteases and serine protease inhibitors plays a key role in the sodium handling in the kidney. Camostat mesilate (CM), a synthetic serine protease inhibitor, reduced prostasin activity and subsequently decreased ENaC current. Oral administration of CM to Dahl salt-sensitive rats resulted in a significant decrease in blood pressure with an elevation of the urinary sodium/potassium ratio. These findings suggest that synthetic serine protease inhibitors such as CM might represent a new class of antihypertensive drugs in patients with salt-sensitive hypertension.

Pancreatic Regeneration by Long-Term Oral Administration of a Protease Inhibitor in a Rat Model of Chronic Pancreatitis

Background/Aims: We previously reported early-stage pancreatic histological features in an experimental chronic pancreatitis model, prepared by injecting oleic acid into the pancreatic duct. In this model, on day 2 after injection, reactive fibroblasts in association with inflammation appeared in the stroma, however, myofibroblasts, which were activated fibroblasts, were not detected. On day 4, myofibroblasts appeared in the stroma, and the lobular architecture was destroyed. On day 28, pancreatic acinar cells were completely necrotized and exfoliated, and the stroma was replaced by collagen fibers. On the other hand, it is known that the long-term oral administration of a protease inhibitor (PI) to rats causes pancreatic enlargement as a trophic effect. In this study, we investigated the trophic effect of an oral PI on the rat pancreas. And we examined the trophic effect on pancreatic regeneration in a pancreatic fibrosis model. Subjects: Male Wistar rats weighing about 200 g were used. ONO-3403 (Ono Pharmaceutical Co., Ltd., Osaka, Japan) was used as a potent oral PI. The PI action of ONO-3403 is five-fold stronger than that of camostat mesilate, the only oral PI now used clinically in Japan. Methods: (1) Normal rats were fed with standard feed or 0.1% ONO-3403-containing feed. The pancreases were removed on day 7, 14, 25. Pancreatic amylase, protein, and DNA contents, as well as pancreatic wet weights, were measured. (2) The abdominal cavity of rats was opened, 100 μl of oleic acid, a cytotoxic agent, was injected into the pancreatic duct, and the cavity was closed. Administration of 0.1% ONO-3403-containing feed was started from day 2 (A gourp) or from day 4 (B group) after injection. On day 28, the pancreases were removed and examined. Results: (1) On day 25 after ONO-3403 administration, the pancreatic wet weights increased over time by about 2.2 times. The amylase, protein, and DNA contents increased by about 3.7, 2.9, and 1.8 times, respectively. The increased protein content suggestive of “hypertrophy” was more marked than the increased DNA content suggestive of “hyperplasia”. (2) In the A group on day 28, pancreatic histological features showed the inhibition of pancreatic fibrosis and regeneration of pancreatic acinar cells. In addition, the pancreatic wet weights and contents similarly increased. However, in the B group on day 28, no improvement of pancreatic fibrosis was found. Conclusions: (1) The long-term oral administration of a potent PI showed marked trophic effects on the rat pancreas, however, the trophic effects contributed to hypertrophy rather than hyperplasia. (2) In a pancreatic fibrosis model, the long-term oral administration of a potent PI, from an early stage when no myofibroblasts appeared in the pancreatic stoma, also showed pancreatic trophic effects and led to the regeneration of pancreatic acinar cells.

Effects of proteolytic enzyme inhibitors as absorption enhancers on the transdermal iontophoretic delivery of calcitonin in rats.

The effects of proteolytic enzyme inhibitors, aprotinin, soybean trypsin inhibitor and camostat mesilate as absorption enhancers on the transdermal iontophoretic delivery of salmon calcitonin (SCT) have been examined in rats. The dermal absorption of SCT was evaluated with hypocalcaemic effect. Application of SCT (12.5 int. units/rat) onto abdominal skin did not produce any hypocalcaemic effect. This produced a small hypocalcaemic effect with cationic iontophoresis (drug phase, anode; reference phase, cathode; high frequency pulses of 1 V at 10 kHz, 2h). Furthermore, camostat mesilate (1 mM) and aprotinin (10(6) int. units mL-1) enhanced the hypocalcaemic effects on the application of SCT with iontophoresis. These hypocalcaemic effects were highest with the pH 4.0 preparation compared with those of the pH 5.5, pH 7.0 and pH 8.0 preparations. However, soybean trypsin inhibitor did not change the hypocalcaemic effects. This was because the soybean trypsin inhibitor is a relatively high molecular weight peptide (mol. wt 8000) and an anion at used pH, and therefore was not absorbed through rat skins with cation iontophoresis.

Use of protease inhibitors to improve calcitonin absorption from the small and large intestine in rats.

The objective of this study was to examine the effects of protease inhibitors on the absorption of calcitonin from different regions of the intestine in rats. The absorption experiments were investigated by in-situ use of closed intestinal loops in rats and stability of calcitonin was examined in mucosal homogenates and intestinal fluids. The intestinal absorption of calcitonin was evaluated by measurement of its hypocalcaemic effect. No substantial hypocalcaemic response was observed when calcitonin was administered into the jejunum or colon. A slight hypocalcaemic effect was observed after administration of calcitonin into the ileum. Of the co-administered protease inhibitors, bacitracin (20mM) strongly promoted calcitonin absorption from the jejunum, ileum and colon. A significant hypocalcaemic effect was also obtained after intestinal administration of calcitonin with soybean trypsin inhibitor (10mgmL(-1)), camostat mesylate (20mM) or aprotinin (2mgmL(-1)). In the stability experiment, bacitracin reduced the degradation of calcitonin in the different intestinal homogenates. Soybean trypsin inhibitor significantly reduced the degradation of calcitonin in the fluids of the small intestine. We also examined the different endopeptidases in gut luminal fluids and the different exopeptidases in gut mucosal homogenates of rats. The ranking order for the total endopeptidase activity of the intestinal fluids was jejunum > ileum > colon. That for total exopeptidase activity of the intestinal mucosa was jejunum > ileum > colon. These results suggest that endo- and exopeptidases might be responsible for the hydrolysis of calcitonin and that protease inhibitors might usefully improve absorption of calcitonin to the systemic circulation from the large intestine.

Su1372 Efficacy of Camostat Mesilate Against Dyspepsia Associated With Non-Alcoholic Mild Pancreatic Disease Based on the Rosemont Criteria

The management of dyspepsia, which is defined as chronic or recurrent pain or discomfort centered in the upper abdomen, remains challenging. The aim of the present study was to examine the potential efficacy of camostat mesilate, a protease inhibitor, against dyspepsia associated with non-alcoholic mild pancreatic disease.

The short term satiety peptide cholecystokinin reduces meal size and prolongs intermeal interval

Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK 1 receptor. However, the literature lacks meal pattern analysis and an appropriate dose–response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800 mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1 nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800 mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose–response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.

A protease inhibitor against acute stress-induced visceral hypersensitivity and paracellular permeability in rats

In the present study, we investigated the effects of camostat mesilate (CM), a synthetic protease inhibitor, on visceral sensitivity and paracellular permeability induced by the acute restraint stress. We also explored the possible mechanisms underlying these effects. The acute restraint stress was induced by wrapping the fore shoulders, upper forelimbs and thoracic trunk of Sprague–Dawley rats for 2 h. Either CM (30, 100 and 300 mg/kg) or saline was intragastrically administrated to the rats 30 min before the acute restraint stress. Visceral perception was quantified as visceral motor response with an electromyography in a subset of rats. Paracellular permeability was determined in another subset of rats. We found that the visceral sensitivity and paracellular permeability were significantly reduced in the CM-treated rats. Moreover, the fecal protease activity was decreased in the CM-treated rats. The ZO-1 protein expression was markedly reduced by the stress treatment, but this decrease was suppressed by CM administration. Our data indicated that CM could efficiently inhibit visceral sensitivity and paracellular permeability induced by the acute restraint stress in rats. Therefore, CM might be an effective drug for the treatment of irritable bowel syndrome.

A Case of Eruption Due to Camostat Mesylate

A Case of Eruption Due to Camostat Mesylate

Preparation and characterization of biodegradable or enteric-coated microspheres containing the protease inhibitor camostat

We have produced biodegradable or enteric-coated microspheres containing camostat mesylate, a protease inhibitor, using a water-oil-water emulsion solvent evaporation method. The characteristics of the microspheres were determined. When polylactic acid, a biodegradable polymer, was used as a wall material, the optimized microsphere obtained showed a loading efficiency of almost 95% and had a mean diameter of 30 microm. This microsphere showed a sustained-release profile, with nearly 25% of drug being released at seven days in a dissolution test. When hypromellose acetate succinate (AS-HG type, with a high content of succinyl group) was used as an enteric wall material, optimized microspheres showed a loading efficiency of almost 80%. In this case, pH 3.0 citrate buffer was used as an internal aqueous phase, and citrate buffer containing 0.5% polyvinylalcohol was used as the external aqueous phase. These microspheres showed a rapid release profile in pH 6.8 buffer, whereas the release was extremely slow in pH 1.2 buffer. Hypromellose acetate succinate microspheres were also prepared containing 10% (w/w) N-benzoyl-dl-arg-4-nitroanilide as a model substrate for trypsin, with or without 5% (w/w) camostat. These microspheres were incubated in pH 6 or 7 buffer containing trypsin at 37 degrees C. When camostat was included in the microspheres, the substrate was protected from attack by trypsin, while in the absence of camostat, the released substrate was immediately attacked by trypsin to produce the degradation product N-benzoyl-dl-arginine.

Refractory Chronic Urticaria Treated Effectively with the Protease Inhibitors, Nafamostat Mesilate and Camostat Mesilate

Chronic urticaria (CU) is characterized by the sponta-neous recurrent appearance of wheals and pruritus, which seriously impairs patients’ quality of life (QoL) (1, 2). Patients with refractory CU are treated with leukotriene receptor antagonists, glucocorticoids and/or other im-munomodulatory agents in addition to antihistamines (3, 4). Nevertheless, there remain a number of patients who are unsatisfactorily treated even with these medications, either due to insufficient efficacy or to the occurrence of side-effects. Nafamostat mesilate (NM) and camostat mesilate (CM) are newly synthesized protease inhibitors. They inhibit broad serine proteases, including trypsin, kallikrein, complements (C1r

Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

ONO 3403, a synthetic serine protease inhibitor, inhibits lipopolysaccharide-induced tumor necrosis factor-α and nitric oxide production and protects mice from lethal endotoxic shock

ONO 3403, a new synthetic serine protease inhibitor, is a derivative of camostat mesilate and has a higher protease-inhibitory activity. The effect of ONO 3403 on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and nitric oxide (NO) production in RAW 264.7 macrophage-like cells was examined. ONO 3403 significantly inhibited LPS-induced TNF-α production at a lower concentration than camostat mesilate. It also inhibited LPS-induced NO production. Their inhibition was responsible for the reduced mRNA expression of TNF-α and inducible NO synthase. In LPS-stimulated cells, ONO 3403 prevented the augmentation of MyD88 expression and inhibited the phosphorylation of IκB-α, stress-activated protein kinase (SAPK) and IRF-3, and the production of interferon-β. ONO 3403 abolished the elevation of the extracellular serine protease activity in response to LPS. Further, it reduced the circulating TNF-α level, hepatic injury and mortality in mice receiving an injection of D-galactosamine and LPS. ONO 3403 was suggested to inhibit LPS-induced inflammatory responses via inactivation of MyD88-dependent and independent pathways.

Preparation and evaluation of an oral delivery system for time-dependent colon release of insulin and selected protease inhibitor and absorption enhancer compounds

The aim of this work was to prepare and evaluate an oral dosage form intended for time-dependent colon delivery of insulin along with a selected protease inhibitor (camostat mesilate) and absorption enhancer (sodium glycocholate). A previously described release platform, which had proven potentially suitable for the protein delivery, was exploited. Insulin compatibility with the above-mentioned adjuvants was preliminarily evaluated. For this purpose, the drug and its main degradation products were assayed by HPLC in insulin powder mixtures with camostat mesilate and/or sodium glycocholate stored 12 months at 4°C. No significant decrease in protein content or increase in degradation product percentages beyond Eur. Ph. 6th Ed. limits was highlighted. Moreover, calorimetric studies performed on physical and compacted binary insulin mixtures with camostat mesilate and sodium glycocholate showed that the thermal behavior of both adjuvants was unchanged. Subsequently, tablet cores with differing compositions were prepared and spray-coated with an aqueous HPMC solution in order to obtain pulsatile delivery systems. The coated units were demonstrated to concurrently release the drug and the adjuvants in a prompt and quantitative mode after consistent lag times. Based on these results, the device was proven a potential candidate for colon delivery of insulin and the selected adjuvants. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4661–4669, 2009